Cell maintainer for autologous cell therapy production

ABSTRACT

In some aspects, the invention relates to automated cell culture incubators and their methods of use. In one aspect, the disclosure provides cell culture incubators having an airlock chamber, a storage chamber and/or an internal chamber. In some aspects, the disclosure provides methods for producing autologous mammalian cell cultures.

RELATED APPLICATIONS

This Application claims the benefit under 35 U.S.C. 119(e) of U.S.provisional application U.S. Ser. No. 62/141,196, filed Mar. 31, 2015,and entitled “Cell Maintainer For Autologous Cell Therapy Production”,the entire contents of which are incorporated by reference herein.

FIELD

Aspects relate to automated cell culture incubators and to methods forusing such incubators. Some aspects relate to methods for producingautologous mammalian cell cultures.

BACKGROUND

Autologous cell therapy is a personalized medicine technique in whichhuman cells are implanted, transplanted, infused, or transferred backinto the individual from whom the cells or tissue were originallyrecovered. For example, chondrocytes may be isolated from a patienthaving a cartilage injury, expanded in a culture system and thenimplanted back into the patient for the purposes of alleviating jointpain associated with the cartilage injury. Other examples of autologouscell therapies include autologous dendritic cells, mesenchymal stromalcells and T lymphocytes. Autologous cell therapies have many advantagesincluding immediate donor availability, reduced cell or tissuerejection, and reduced graft-versus-host disease. Additionally, theautologous nature of the cells means there is no need for HLA matchingand immunosuppression of the cell recipient. Although there are manytherapeutic advantages of autologous cell therapy, several challenges inthe manufacturing and production of autologous cell cultures imposebarriers to commercial success.

SUMMARY

One challenge for the manufacturing autologous cell therapies is“scale-out”, or the ability to simultaneously produce multiple batchesof autologous cell cultures from different donors. Strict asepticconditions must be maintained in order to prevent cross-contaminationbetween autologous cultures from different patients. Some currently usedmethods and devices for autologous cell culture rely on manual culturetechniques, which can introduce contaminants to cultures and exposecultures to non-aseptic conditions and/or variations of the physicalenvironment (e.g., changes in temperature, humidity, etc., or anycombination thereof). Other cell culture apparatus do not provide theability to culture cells from multiple donors with minimal risk ofcross-contamination. Accordingly, new cell culture systems and methodsare provided herein that permit remote maintenance and significantscale-out capabilities of multiple cell cultures while maintainingstringent aseptic conditions.

In some aspects, this document provides a method for producing amammalian cell culture that include: (a) introducing a mammalian cellsample into a cell culture vessel in the presence of growth media,wherein the vessel includes a passage configured to permit materials tobe aseptically transferred into or out of the vessel; and, (b) expandingthe cell sample in an incubator into a mammalian cell culture, whereinthe incubator comprises a sterile inner growth chamber.

In some embodiments, the cell sample is introduced into the cell culturevessel through the passage. In some embodiments, the passage of thevessel is covered by a gas-permeable membrane. In some embodiments, amembrane on a culture vessel provides a one-way valve or “environmental”interface through which the gaseous environment may be controlled withinthe closed, autologous culture vessel. In some embodiments, multipleclosed systems (e.g., within a single, temperature controlledenvironment) may be provided in the form of a plurality of autologousculture vessels. In some embodiments, an overall incubator environmentmaintains the environmental temperature of the collective system. Insome embodiments, the internal gaseous environment of each closed vesselis maintained through an interface (e.g., a plumbing interface) whichcontains a set of membrane “valves” for gas exchange. In suchembodiments, this configuration facilitates control, monitoring anddocumentation of independent autologous cultures.

In some embodiments, the method further comprises asepticallyintroducing growth media to the cell culture through the passage in thevessel.

In some embodiments, the method further comprises asepticallyintroducing a biological material to the cell culture through theopening in the vessel. In some embodiments, the biological material is acell growth factor. In some embodiments, the biological material is anucleic acid molecule. In some embodiments, the nucleic acid molecule isa nucleic acid vector. In some embodiments, the nucleic acid vector is atransfection vector. In some embodiments, the nucleic acid vector is atransduction vector. In some embodiments, the nucleic acid vectorcomprises transgenic material. In some embodiments, the biologicalmaterial is an enzyme (e.g., a nuclease, a ligase, a polymerase, orother enzyme that modifies a nucleic acid). In some embodiments, thebiological material comprises a mixture of nucleic acid modifyingenzyme(s) and one or more nucleic acids.

In some embodiments, the methods further comprise aseptically monitoringconditions of the growth media. In some embodiments, the incubator ismaintained at a constant temperature range. In some embodiments, theincubator is maintained at about 37 degrees Celsius.

In some embodiments, the methods further comprise aseptically monitoringconditions of the cell.

In some embodiments, the methods further comprise aseptically addingreagents to control the chemical composition of the growth media (e.g.,the pH, the concentration of glucose, the concentration of lactate, theconcentration of other small molecules, or the overall osmolality of thegrowth media).

In some embodiments, the methods further comprise aseptically imagingthe cell culture. In some embodiments, the method further comprisesfiltering the cell culture. In some embodiments, the method furthercomprises aseptically removing an aliquot of the cell culture. In someembodiments, the incubator is a cell culture system as described herein.

In some aspects, this document provides a cell culture system comprisingan incubator cabinet comprising: a transfer chamber; one or moreinternal chambers; an external door opening from an external environmentto the transfer chamber; a first internal door opening from the transferchamber to a first internal chamber; a second internal door opening fromthe transfer chamber to a second internal chamber; and a transfer devicefor moving one or more items between the transfer chamber and the firstinternal chamber, and/or between the transfer chamber and the secondinternal chamber and/or between the second internal chamber and thefirst internal chamber.

In some embodiments, the cell culture system further comprises asterilization medium supply (e.g., an ozone generator) coupled to apump, wherein the sterilization medium supply (e.g., the ozonegenerator) is configured for supplying sterilization medium (e.g., ozonegas) to the transfer chamber. In some embodiments, the sterilizationmedium supply (e.g., ozone generator) is configured for supplyingsterilization medium (e.g., ozone gas) to the one or more internalchambers.

In some embodiments, the external door forms a substantially gas-tightseal when closed. In some embodiments, the first internal door and thesecond internal door each forms a substantially gas-tight seal whenclosed.

In some embodiments, the pump is configured to remove sterilizationmedium from the transfer chamber and/or one or more internal chambers.

In some embodiments, the storage chamber comprises a storage location.In some embodiments, the storage location is configured to hold aplurality of cell culture vessels. In some embodiments, each vessel ofthe plurality of cell culture vessels contains cells from a differentpatient. In some embodiments, each vessel of the plurality of cellculture vessels is tagged with a unique barcode.

In some embodiments, the internal chamber comprises an imager and animaging location. In some embodiments, the imager is a holographicimager. In some embodiments, the imager is a microscope, such as abright-field microscope or a fluorescence microscope. In someembodiments, the cell culture system further comprises a controller forthe imager.

In some embodiments, the internal chamber comprises a manipulator and amanipulation location. In some embodiments, the manipulator is a cellpicker. In some embodiments, the manipulator comprises a fluid handlingsystem.

In some embodiments, the cell culture system further comprises acontroller for the manipulator.

In some embodiments, the internal chamber comprises a fluid storagelocation. In some embodiments, the internal chamber comprises a cellsorting or cell isolation apparatus. In some embodiments, the cellsorting or cell isolation apparatus is a centrifuge. In someembodiments, the cell sorting or cell isolation apparatus is aFluorescence-Activated Cell Sorting (FACS) machine. In some embodiments,the internal chamber comprises a microfluidic device for imaging and/ormanipulating individual cells.

In some embodiments, the transfer device comprises one or more roboticelements. In some embodiments, the cell culture system further comprisesa controller for the transfer device. In some embodiments, thecontroller for the imager, the controller for the manipulator, and/orthe controller for the transfer device are external to the incubatorcabinet. In some embodiments, the controller for the imager, thecontroller for the manipulator and/or the controller for the transferdevice comprises a single processor. In some embodiments, the controllerfor the imager, the controller for the manipulator, and/or thecontroller for the transfer device comprise a computer. In someembodiments, a single computer controls the imager, the manipulator,and/or the transfer device.

In some embodiments, the cell culture system further comprises a barcodescanner. In some embodiments, the barcode scanner is connected to acomputer, wherein the computer is external to the incubator cabinet.

In some aspects, this document provides a cell culture system comprisingtwo or more cell culture vessels, wherein each vessel comprises cellsfrom a different patient and wherein each vessel comprises a passageconfigured to permit materials to be aseptically passed into or out fromthe vessel.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures may be represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. Various embodiments of the invention will now be described, byway of example, with reference to the accompanying drawings, in which:

FIG. 1 is a schematic of an illustrative embodiment of a cell culturesystem comprising an incubator cabinet comprising an internal chambercontaining a plurality of cell culture vessels. Each cell culture vesselcontains a cell culture from a different subject;

FIGS. 2A-2B are schematics of illustrative embodiments of cell culturesystems; FIG. 2A shows a schematic of a cell culture system comprisingan incubator cabinet comprising a transfer chamber and an internalchamber; FIG. 2B shows a schematic of a cell culture incubatorcomprising an incubator cabinet comprising a transfer chamber, aninternal chamber containing a plurality of cell culture vessels, anozone generator, and a pump;

FIG. 3 is a schematic of an illustrative embodiment of a cell culturesystem comprising an incubator cabinet comprising a transfer cabinet, afirst internal chamber containing a plurality of cell culture vessels,and a second internal chamber comprising an imager and a manipulator;

FIG. 4 is a schematic of an illustrative embodiment of a cell culturesystem comprising an incubator cabinet comprising an internal chamber, aplurality of cell culture vessels, and an imager.

DETAILED DESCRIPTION

Currently used cell culture incubators impose barriers to the success ofautologous cell culture. For example, many cell culture incubatorsrequire the removal of cell culture vessels and their subsequent manualhandling. Removal of cultured cells from the protected environmentprovided by an incubator increases exposure of the culture to potentialcontaminants, including cross-contamination from other autologous cellcultures, and/or variations of the physical environment (e.g., changesin temperature, humidity, etc., or any combination thereof).Furthermore, manual handling of cultures by human operators introducesthe possibility of contamination introduced by human error, such asimproper sterile technique. The methods and apparatus for remotemaintenance (e.g., with minimal human handling) of autologous cellcultures in this document overcome these barriers and issues. Thisdocument is based, in part, on development of cell culture vessels thathave the capability to allow aseptic passage of materials into and outof the cell culture vessel and to allow the simultaneous production of aplurality of cell cultures from different subjects while minimizing riskof cross-contamination between the cultures and the exposure of theculture to a non-aseptic and/or non-controlled physically environment.

Cell Culture Methods

In one aspect, this document relates to a method for producing amammalian cell culture including the steps of introducing a mammaliancell sample into a cell culture vessel in the presence of growth media,wherein the vessel has a passage configured to permit materials to beaseptically passed into or out from the vessel; and expanding the cellsample in an incubator into a mammalian cell culture, wherein theincubator includes a sterile internal chamber.

As used herein, “cell culture” refers to a procedure for maintainingand/or growing cells under controlled conditions (e.g., ex vivo). Insome embodiments, cells are cultured under conditions to promote cellgrowth and replication, conditions to promote expression of arecombinant product, conditions to promote differentiation (e.g., intoone or more tissue specific cell types), or a combination of two or morethereof.

As used herein, the term “mammalian cell sample” refers to any cellobtained from a mammalian subject. Non-limiting examples of mammaliansubjects include humans, non-human primates, mice, rats, horses, dogs,cats, and guinea pigs. In some embodiments, the mammalian cell sample isobtained from a human.

In some embodiments, a cell sample is isolated from a tissue or organ(e.g., a human tissue or organ), including but not limited to solidtissues and organs. In some embodiments, cell samples can be isolatedfrom placenta, umbilical cord, bone marrow, liver, blood, including cordblood, or any other suitable tissue. In some embodiments,patient-specific cell samples are isolated from a patient for culture(e.g., for cell expansion and optionally differentiation) and subsequentre-implantation into the same patient or into a different patient. Insome embodiments, cells grown in an incubators described herein may beused for allogenic or autogeneic therapy. In some embodiments, cellsgrown in the incubators disclosed herein may be genetically modified,expanded and reintroduced into a patient for the purpose of providing animmunotherapy (e.g., chimeric antigen receptor therapy (CAR-T), ordelivery of CRISPR/Cas modified cells).

In some embodiments, cells are isolated from tissues or biologicalsamples for ex vivo culture in an incubator described herein. In someembodiments, cells (e.g., white blood cells) are isolated from blood. Insome embodiments, cells are released from tissues or biological samplesusing physical and/or enzymatic disruption. In some embodiments, one ormore enzymes such as collagenase, trypsin, or proteinase are used todigest the extracellular matrix. In some embodiments, tissue orbiological samples are placed in culture medium (e.g., with or withoutphysical or enzymatic disruption), and cells that are released and thatgrow in the culture medium can be isolated for further culture.

The methods described herein are suitable for culturing a variety ofmammalian cell types. In some embodiments, the mammalian cell sample isa cell useful for autologous cell therapy. As used herein, the term“autologous cell therapy” refers to the implantation, transplantation,infusion, or transfer of cultured cells back into the individual fromwhom the cells were obtained. For example, immune cells may be obtainedfrom a subject having cancer, expanded into a cell culture, primed withan antigen against the cancer, and reintroduced into the patient inorder to boost the subject's immune response. Examples of cells that areuseful for autologous culture include but are not limited to, stem cells(e.g., hematopoietic stem cells, somatic stem cells, totipotent stemcells, pluripotent stem cells, fetal stem cells, embryonic stem cells,mesenchymal stem cells, and induced pluripotent stem cells), progenitorcells (e.g., satellite cells, neural progenitor cells, bone marrowstromal cells, pancreatic progenitor cells, angioblasts and endothelialprogenitor cells), immune cells (e.g., T-lymphocytes, dendritic cells)and differentiated cells (epithelial cells, cardiomyocytes, fibroblasts,and chondrocytes).

As described herein passages can be configured to permit aseptictransfer of materials into and out of a vessel in a manner that isuseful for minimizing cross-contamination during the simultaneousculture of mammalian cells from different subjects. In some aspects, themethods provided herein relate to using a cell culture vessel, whereinthe vessel includes a passage configured to permit materials to beaseptically transferred into or out from the vessel. As used herein, theterm “passage” refers to a conduit that allows the movement of materialsinto or out of the vessel. For example the vessel may be an open-endedtube that is sealed at its opening by a pierceable, self-sealingmembrane. In some embodiments, the membrane is a gas-permeable membrane.In some embodiments, the passage is a sterile, disposable tube in fluidcommunication with a sealed, gas-permeable vessel. In some embodiments,the passage is a network of microfluidic channels, having inlet andoutlet ports capable of being sterilized, that are integrated into asealed culture vessel.

As used herein, the term “aseptically transferred” refers to themovement of material from one location to another without theintroduction of contaminants. For example, it may be desirable thatculture media is aseptically transferred from a storage container to acell culture vessel because non-aseptic transfer of the media couldintroduce pathogens or other contaminants into the cell culture.Contaminants include but are not limited to bacteria (e.g. pathogenicbacteria and non-pathogenic bacteria), viruses (e.g. pathogenic virusesand non-pathogenic viruses), molds, spores, and dust. In someembodiments, a contaminant is a cell. For example, when cells obtainedfrom different subjects are simultaneously cultured, aseptic transfer ofmaterials is required so that cross-contamination (i.e., theintroduction of a cell or media from one culture to a different culture)does not occur.

Aseptic techniques can be used to prevent or minimize contamination ofcell cultures during growth and manipulation. In some embodimentsequipment (e.g., pipettes, fluid handling devices, manipulating devices,other automated or robotic devices, etc.) that is used for cell cultureis sterilized using an appropriate technique. Non-limiting techniquesinclude heat exposure (e.g., autoclaving), surface disinfection (e.g.,using alcohol, bleach, or other disinfectant), irradiation, and/orexposure to a disinfectant gas (e.g., ozone, hydrogen peroxide, etc.) asdescribed herein. In some embodiments, media is sterilized using anappropriate technique. Non-limiting techniques include heat exposure(e.g., autoclaving), antimicrobial/antiviral treatment, filtration,and/or irradiation.

In some embodiments, manipulations of cell cultures are performed underaseptic conditions, for example, in an environment (e.g., within anincubator chamber) that has been disinfected and in which the air hasbeen filtered to remove potential contaminants.

In some embodiments, cell cultures are grown and maintained underGMP-compliant conditions, including those that include usingGMP-compliant media or GMP-compliant liquid handling equipment. In somecases, cell cultures are grown and maintained by performing methods inconjunction with standard operation procedures (SOPs).

In some embodiments, cell cultures can be monitored and/or evaluated todetect contamination. In some embodiments, contamination by cells from adifferent type of organism can be detected. In some embodiments,contamination of a mammalian cell culture by mycoplasma, bacteria,yeast, or viruses can be detected using any suitable technique. In someembodiments, cell culture contamination can be detected by assaying forchanges or for rates of change of one or more culture properties such aspH, turbidity, etc., that are characteristic of contamination (e.g., bybacteria or yeast) and not characteristic of the cells (e.g., mammaliancells) being grown in culture. In some embodiments, one or moremolecular detection assays (e.g., PCR, ELISA, RNA labeling, or otherenzymatic techniques) or cell-based assays can be used to detectcontamination (e.g., mycoplasma, bacterial, yeast, viral, or othercontamination).

In some embodiments, cell cultures can be monitored and/or evaluated todetect contamination with cells of similar types (e.g., a human cellline contaminated by different human cells or by different mammaliancells). In some embodiments, cell cultures and their potentialcontamination can be evaluated using DNA sequencing or DNAfingerprinting (e.g., short tandem repeat—STR—fingerprinting), isoenzymeanalysis, human lymphocyte antigen (HLA) typing, chromosomal analysis,karyotyping, cell morphology, or other techniques.

In some embodiments, cells produced using the incubators or methodsdescribed herein can be frozen to preserve them for later use and/or fortransport. In some embodiments, cells are mixed with a cryopreservationcomposition after growth and/or differentiation and prior to freezing. Acryopreservation composition can be added to a cell culture vessel, orcells can be transferred from a cell culture vessel to acryopreservation vessel along with a cryopreservation composition.Non-limiting examples of cryoprotectants that can be included in acryopreservation composition include DMSO, glycerol, PEG, sucrose,trehalose, and dextrose. In some embodiments, a freezer may be providedas a component of an incubator to facilitate freezing of cells isolatedfrom cell cultures. For example, one or more freezers may be located inan internal chamber and/or integrated into the incubator cabinet (e.g.,into a wall of the incubator cabinet).

As used herein, a “cell culture vessel” is a device including a housingand one or more chambers for culturing cells. In some embodiments, thehousing is a frame. The frame may be coupled to a lid. The one or morechambers may include cell culturing media including one or moremembranes. In some embodiments, a cell culture vessel may includenutrients for promoting the growth of cells. In certain embodiments, acell culture vessel may entirely enclose one or more cells or groupsthereof. The housing of a cell culture vessel may include one or morepores or openings to permit the transfer of gases between a cell culturevessel and its surrounding environment. In certain embodiments, a cellculture vessel includes a transparent or optically clear window. Forexample, a lid coupled to the housing of a cell culture vessel mayinclude an optically clear portion for viewing cells e.g., with amicroscope or other imager.

Various types of cell culture vessels can be designed as describedherein. Cell culture vessels can be made from any non-reactivebiocompatible material, such as glass, plastic or silicone. Generally,cell culture vessels are formed into bottles, flasks, vials, bags, tubesor culture plates. In some embodiments, the cell culture vessel is avial. In some embodiments, the cell culture vessel is a bottle or flask.In some embodiments, the cell culture vessel is a culture plate. In someembodiments, the plate is a cell culture dish. In some embodiments, theplate is a multi-well culture plate. Generally multi-well plates includean array of 96, 384 or 1536 wells. In some embodiments, a cell culturevessel includes one or more portions that are substantiallynon-reflective. In some embodiments, the cell culture vessel isbarcoded. In some embodiments, an incubator includes a barcode reader.

In some embodiments, cell culture vessels may be pre-kitted with one ormore reagents desired for a particular purpose, e.g., for growing cells,for differentiating cells, for subjecting cells to a particular assaycondition, etc. In some embodiments, pre-kitted cell culture vesselscontain reagents useful for performing a particular experiment (e.g.,cell growth media, growth factors, selection agents, labeling agents,etc.) on a cell culture, in advance of the experiment. Pre-kitted cellculture vessels may facilitate experimental protocols by providing cellculture-ready vessels that do not require the addition of reagents. Forexample, progenitor cells from a patient may be added to a cell culturevessel pre-kitted with reagents for cell differentiation for the purposeof expanding a population of differentiated cells for autologous celltherapy. Pre-kitted cell culture vessels can be stored at anyappropriate temperature, which is determined by the recommended storageparameters of the reagents within the pre-kitted cell culture vessel. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about −80° C. and about 37° C. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about −80° C. and about −20° C. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about −20° C. and about 4° C. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about 4° C. and about 37° C. Insome embodiments, pre-kitted cell culture vessels are disposable. Insome embodiments, pre-kitted cell culture vessels are reusable and/orrefillable.

In some embodiments, cell culture vessels are configured for culturingcells in suspension. In some embodiments, cell culture vessels areconfigured for culturing adherent cells. In some embodiments, cellculture vessels are configured for 2D or 3D cell culture. In someembodiments, cell culture vessels include one or more surfaces ormicro-carriers to support cell growth. In some embodiments, these arecoated with extracellular matrix components (e.g., collagen, fibrinand/or laminin components) to increase adhesion properties and/or toprovide other signals needed for growth and differentiation. In someembodiments, cell culture vessels include one or more synthetichydrogels such as polyacrylamide or polyethylene glycol (PEG) gels tosupport cell growth. In some embodiments, cell culture vessels include asolid support with embedded nutrients (e.g., a gel or agar, for examplefor certain bacterial or yeast cultures). In some embodiments, cellculture vessels include a liquid culture medium.

In some embodiments, growth media is aseptically introduced into thecell culture vessel. As used herein, the term “growth media” refers to amedium for culturing cells containing nutrients that maintain cellviability and support proliferation. In some cases various parametersand conditions can be used for culturing cells. The growth media maycontain any of the following nutrients in appropriate amounts andcombination: salt(s), buffer(s), amino acids, glucose or other sugar(s),antibiotics, serum or serum replacement, and other components such aspeptide growth factors, etc. Growth media are known in the art and maybe classified as natural or artificial media. Examples of cell culturemedia include but are not limited to Minimum Essential Medium (MEM),Dulbecco's Modified Eagle's Medium (DMEM), and Roswell Park MemorialInstitute Medium (RPMI). An appropriate medium for culturing the cellmay be selected.

In some embodiments, cells are cultured in one of any suitable culturemedia. Different culture media having different ranges of pH, glucoseconcentration, growth factors, and other supplements can be used fordifferent cell types or for different applications. In some embodiments,custom cell culture media or commercially available cell culture mediasuch as Dulbecco's Modified Eagle Medium, Minimum Essential Medium, RPMImedium, HA or HAT medium, or other media available from LifeTechnologies or other commercial sources can be used. In someembodiments, cell culture media include serum (e.g., fetal bovine serum,bovine calf serum, equine serum, porcine serum, or other serum). In someembodiments, cell culture media are serum-free. In some embodiments,cell culture media include human platelet lysate (hPL). In someembodiments, cell culture media include one or more antibiotics (e.g.,actinomycin D, ampicillin, carbenicillin, cefotaxime, fosmidomycin,gentamycin, kanamycin, neomycin, penicillin, penicillin streptomycin,polymyxin B, streptomycin, tetracycline, or any other suitableantibiotic or any combination of two or more thereof). In someembodiments, cell culture media include one or more salts (e.g.,balanced salts, calcium chloride, sodium chloride, potassium chloride,magnesium chloride, etc.). In some embodiments, cell culture mediainclude sodium bicarbonate. In some embodiments, cell culture mediainclude one or more buffers (e.g., HEPES or other suitable buffer). Insome embodiments, one or more supplements are included. Non-limitingexamples of supplements include reducing agents (e.g.,2-mercaptoethanol), amino acids, cholesterol supplements, vitamins,transferrin, surfactants (e.g., non-ionic surfactants), CHO supplements,primary cell supplements, yeast solutions, or any combination of two ormore thereof. In some embodiments, one or more growth or differentiationfactors are added to cell culture media. Growth or differentiationfactors (e.g., WNT-family proteins, BMP-family proteins, IGF-familyproteins, etc.) can be added individually or in combination, e.g., as adifferentiation cocktail including different factors that bring aboutdifferentiation toward a particular lineage. Growth or differentiationfactors and other aspects of a liquid media can be added using automatedliquid handlers integrated as part of an incubator provided herein.

In some embodiments, biological material is aseptically introduced intothe cell culture vessel. Examples of biological materials include butare not limited to growth factors, nucleic acids, and expressionvectors. Growth factors are naturally occurring substances thatstimulate cell growth, proliferation, healing and/or differentiation.Generally, growth factors are proteins or steroid hormones. In thecontext of mammalian cell culture, growth factors may be introduced toculture media in order to control the cell cycle or induce proliferationor differentiation of cultured cells. Non-limiting examples of growthfactors include angiopoietin, bone morphogenic proteins (BMPs),epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF),erythropoietin (EPO), fibroblast growth factor (FGF), granulocytecolony-stimulating factor (G-CSF), insulin-like growth factor (IGF),nerve growth factor (NGF), transforming growth factor beta (TGF-β), andvascular endothelial growth factor (VEGF).

In some embodiments, the biological material is a nucleic acid orexpression vector. For example, somatic cells can be “reprogrammed” tobecome induced stem cells via the introduction of genetic materialencoding reprogramming protein factors and microRNA. In someembodiments, the methods provided herein further included asepticintroduction of a nucleic acid or expression vector into the cellculture vessel. In some embodiments, a nucleic acid is introduced to thecell culture. Examples of nucleic acids include DNA, RNA, siRNA, miRNA,ami-RNA, shRNA, and dsRNA. In some embodiments, an expression vector isintroduced into the cell culture vessel. The term “expression vector”refers to an engineered molecule capable of artificially carryingforeign genetic material into another cell and expressing the geneticmaterial in the cell. Expression vectors can generally be classified astransfection vectors and transduction vectors. Transfection vectors(e.g., DNA-based plasmid vectors) are generally used fornon-virally-mediated transfer of genetic material into cells.Transduction vectors (e.g., lentivital vectors, AAV vectors, rAAVvectors, and retroviral vectors) are generally used for virally-mediatedtransfer of genetic material into cells. In some embodiments, theexpression vector includes a transgene. The composition of the transgenesequence of an expression vector will depend upon the use to which theresulting vector will be put. For example, one type of transgenesequence includes a reporter sequence, which upon expression produces adetectable signal. In another example, the transgene encodes atherapeutic protein or therapeutic functional RNA.

In some aspects, this documents relates to methods for monitoring cellsunder controlled conditions (e.g., under aseptic and/or sterileconditions). In some aspects, methods described herein are useful forcell culture (e.g., to grow and maintain cells for recombinant proteinexpression or to grow and/or differentiate cells for therapeuticapplications such as implantation). In some embodiments, the conditions(e.g., environment) inside incubators provided herein are monitored. Insome cases, the temperature, humidity, carbon dioxide, oxygen and othergaseous components inside the incubator may be monitored. In someembodiments, the conditions (e.g., temperature, oxygen, carbon dioxide,and pH) of the growth media are monitored. Growth media conditions canbe monitored directly, via probes and sensors, or indirectly viacolorimetric (e.g., media containing Phenol Red) or imaging techniques(e.g., infrared or thermal imaging). In some embodiments, conditions ofgrowth media and cells are monitored by aseptically removing an aliquotcontains growth media and cells from a culture vessel and analyzing thealiquot at a location external to the culture vessel. In someembodiments, the aliquot is filtered, for example, by centrifugation toseparate the cells from the growth media.

In some embodiments, incubators and methods described herein are used tomonitor or assay culture media for nutrient depletion, changes in pH,changes in temperature, accumulation of apoptotic or necrotic cells,and/or cell density. In some embodiments, incubators and methodsdescribed herein are used to modify or change the culture media orconditions and/or to passage the cell cultures when appropriate. In someembodiments, the methods described herein are automated.

Cell Culture Systems

In some aspects, this document relates to cell culture systems includingan incubator cabinet. As used herein, an “incubator cabinet” is ahousing that includes one or more chambers configured to hold one ormore cell culture vessels. In some embodiments, an incubator cabinetincludes a transfer chamber and an internal chamber, one or both ofwhich are configured to hold one or more cell culture vessels. In someembodiments, an incubator may include one or more other elements such asone or more gas sources (e.g., a gas cylinder or ozone generator),tubing (e.g., to convey one or more liquids or gases such as water,distilled water, deionized water, cell culture medium, air, carbondioxide, ozone, and oxygen), airflow mechanisms (e.g., valves, releasevalves, pinholes, gas regulators, and mass flow regulators), pressuremechanisms (e.g., a pump such as a dry scroll pump, rotary pump,momentum transfer pump, diffusion pump, or diaphragm pump; a suctiontube; a vacuum system; and an air blower), environmental monitors andcontrols (e.g., a gas sensor and/or monitor to sense and/or controlconcentrations of gases such as carbon dioxide, oxygen, and ozone; heatsources or sinks; temperature monitors and controls; humidity monitors;gas scrubbers; air filters; instrumentation for measuring particulatematter; pressure gauges; and flow meters), doors (e.g., openings orpanels), windows (e.g., optical windows made of glass, plastic,composite, or other substantially transparent material for viewing anarea inside the incubator cabinet), ports (e.g., to permit theintroduction or removal of one or more gases or liquids), light sources(e.g., lamps, bulbs, lasers, and diodes), optical elements (e.g.,microscope objectives, mirrors, lenses, filters, apertures, wave plates,windows, polarizers, fibers, beam splitters, and beam combiners),imaging elements (e.g. cameras, barcode readers), electrical elements(e.g., circuits, cables, power cords, and power supplies such asbatteries, generators, and direct or alternating current supplies),computers, mechanical elements (e.g., motors, wheels, gears, roboticelements, and actuators such as pneumatic actuators, electromagneticactuators, motors with cams, piezoelectric actuators, and motors withlead screws), and control elements (e.g., spin-wheels, buttons, keys,toggles, switches, cursors, screws, dials, screens, and touch-screens).In some embodiments, one or more of these other elements are part of theincubator, but are external to the incubator cabinet. In someembodiments, one or more of these other elements are included within theincubator cabinet.

In some embodiments this document relates to incubators and methods forculturing, manipulating, and/or monitoring cells under controlledconditions (e.g., under aseptic and/or sterile conditions). In someembodiments, the cell culture incubators included an incubator cabinethaving an internal chamber for incubation of cells in one or more cellculture vessels. In some cases, in addition to an internal door from thetransfer chamber to the internal chamber, the incubators include atleast one external door (e.g., 1, 2, 3, 4, or more external doors)opening from an external environment directly to the internal chamber,for example, to provide alternative access to the internal chamberduring periods of time when the incubator is not operational, e.g.,during maintenance of the incubator. In some embodiments, incubatorsinclude a storage location within the internal chamber for storing oneor more cell culture vessels.

In some embodiments, incubators or incubator cabinets provided hereinare rectangularly cuboidal in shape. In some embodiments incubators orincubator cabinets provided herein have a rectangular footprint in arange of 1 ft² to 16 ft². In some embodiments incubators or incubatorcabinets provided herein have a rectangular footprint of up to about 1ft², 2 ft², 3 ft², 4 ft², 5 ft², 6 ft², 7 ft², 8 ft², 9 ft², 10 ft², 11ft², 12 ft², 13 ft², 14 ft², 15 ft², or 16 ft². In some embodimentsincubators or incubator cabinets provided herein have a total chambervolume in a range of 1 ft³ to 100 ft³. In some embodiments incubators orincubator cabinets provided herein have a chamber volume of up to about1 ft³, 5 ft³, 10 ft³, 25 ft³, 50 ft³ or 100 ft³. In some embodimentsincubators or incubator cabinets provided herein have a rectangularfootprint in a range of 0.09 m² to 1.78 m². In some embodimentsincubators or incubator cabinets provided herein have a rectangularfootprint of up to about 0.1 m², 0.2 m², 0.3 m², 0.4 m², 0.5 m², 0.6 m²,0.7 m², 0.8 m², 0.9 m², 1.0 m², 1.1 m², 1.2 m², 1.3 m², 1.4 m², 1.5 m²,1.6 m², or 1.7 m². In some embodiments, incubators or incubator cabinetsprovided herein have a total chamber volume in a range of 0.03 m³ to 3m³. In some embodiments incubators or incubator cabinets provided hereinhave a chamber volume of up to about 0.03 m³, 0.1 m³, 0.3 m³, 1 m³, or 3m³.

In some embodiments, an incubator cabinet is single-walled. In someembodiments, an incubator is double-walled. In some embodiments,insulation material is provided between the double walls of an incubatorcabinet to control heat loss from the cabinet and facilitate temperaturecontrol in the cabinet. In some embodiments, the outer wall of anincubator cabinet includes a sheet metal, e.g., a 14-20 gauge coldrolled steel. In some embodiments, an inner wall (e.g., a chambersurface) of an incubator cabinet includes electro-polished stainlesssteel. In some embodiments, an inner wall (e.g., a chamber surface) ofan incubator cabinet includes corrosion resistant materials, such as,titanium, cobalt-chrome, tantalum, platinum, zirconium, niobium,stainless steel, and alloys thereof. However, in some embodiments, achamber surface of an incubator cabinet includes a polymeric materialsuch as polytetrafluoroethylene (PTFE), or a polymeric material knowunder the trade name of Parylene. In some embodiments, a chamber surfacemay have anti-microbial properties, such as copper or silver oranti-microbial compounds incorporated into a polymeric surface coating.

In some embodiments, the incubator includes an airlock arrangement thatmay be used to help decreases exposure of the internal chamber to theexternal environment, or exposure of the external environment to theinternal chamber. For example, an incubator cabinet may include anexternal door leading to a transfer chamber and an internal chamber,wherein the transfer chamber and the internal chamber are physicallyseparated by a wall having an internal door. In some embodiments, toutilize the airlock arrangement, one door is opened at a time. Forexample, an operator may open the external door to gain access to thetransfer chamber. The operator may then insert item(s) such as pipettetips into the transfer chamber. An operator may operate the externaldoor by directly manipulating the door. In some embodiments, an operatormay operate the door indirectly by controlling the operation of the doorremotely, e.g., through the use of automation configured to controlopening and closing of the doors. In some embodiments, the internalchamber door remains closed while the external door is open. In someembodiments, after item(s) are inserted into the transfer chamber, theexternal door is closed (e.g., directly or indirectly by an operator).Once the external door is closed, a sterilization process inside thetransfer chamber is used to sterilize the inserted item(s). Oncesterilization is complete, the internal chamber door is opened, and thesterilized items are moved from the transfer chamber into the internalchamber (e.g., by one or more transfer devices).

In some embodiments, the transfer chamber and/or the internal chambermay have a gas-tight or hermetic seal, e.g., around one or more windowsor doors. In particular embodiments, sealants such as grease and/ormechanical elements such as o-rings, gaskets, septa, KF, LF, QF, quickcoupling, or other sealing mechanisms may be used to establish one ormore gas-tight seals. In some embodiments, grooves, depressions,protrusions, and/or molded plastic elements may facilitate inestablishing one or more gas-tight seals. In some embodiments, anincubator (e.g., an internal chamber, and/or a transfer chamber of anincubator cabinet) includes one or more windows and/or doors, that, whenclosed, are sealed to preserve sterility (e.g., after one or morechambers of the incubator have been sterilized). In some embodiments,each seal of the incubator is air tight up to a threshold level ofpressure (e.g., up to 1 atm). In some embodiments, a gasket is providedto ensure a desired level of sealing capacity. In general, a “gasket” isunderstood as a mechanical seal that fills the space between twoobjects, generally to prevent leakage between the two objects whileunder compression. Gaskets are commonly produced by cutting from sheetmaterials, such as gasket paper, rubber, silicone, metal, cork, felt,neoprene, nitrile rubber, fiberglass, or a plastic polymer (such aspolychlorotrifluoro-ethylene). It is often desirable that a gasket bemade from a material that provides some degree of yielding such that itis able to deform and fill tightly the space it is designed for,including any slight irregularities. In some embodiments, gaskets can beused with an application of sealant directly to the gasket surface tofunction properly. In some embodiments, a gasket material can be aclosed-cell neoprene foam which is non-reactant with carbon dioxide orozone.

Internal Chambers

As used herein, an “internal chamber” is a chamber disposed in anincubator cabinet. An internal chamber may include one or more windows(e.g., optical windows made of glass, plastic, composite, or othersubstantially transparent material for viewing an area inside theincubator cabinet). An internal chamber may include at least one door(e.g., for permitting the transfer of items into or out of the internalchamber). In some embodiments, the at least one door may be disposedbetween the internal chamber and a transfer chamber. In certainembodiments, an interlock may prevent the door from opening at anundesirable time (e.g., when a portion of the incubator cabinet is opento the surrounding environment so that contaminants cannot enter theinternal chamber). An internal chamber may be of any appropriate sizeand geometry. In some embodiments, an incubator cabinet may include morethan one internal chamber. In other embodiments, an internal chamber mayinclude one or more partitions to define different regions of aninternal chamber. One or more internal chambers or partitions thereofmay have different environmental conditions. The environment (e.g., airpressure, gas content, temperature, light, and humidity) inside aninternal chamber may be measured and/or controlled by one or moremeters, monitors, sensors, controls, pumps, valves, apertures, and/orlight sources. In some embodiments, an internal chamber may have agas-tight or hermetic seal, e.g., around one or more windows or doors.In particular embodiments, sealants such as grease and/or mechanicalelements such as o-rings, gaskets, septa, KF, LF, QF, quick coupling, orother sealing mechanisms may be used to establish one or more gas-tightseals. In some embodiments, grooves, depressions, protrusions, and/ormolded plastic elements may facilitate in establishing one or moregas-tight seals.

An internal chamber may be made of any useful material. In someembodiments, an internal chamber may include one or more plastics,polymers, metals, or glasses.

As used herein, a “door” is an element that permits communicationbetween two or more environments or regions when opened and preventscommunication between the two or more environments or regions whenclosed. A door may be of any type, such as a sliding door, pocket door,swinging door, hinged door, revolving door, pivot door, or folding door.The door may be manually, mechanically, or electrically operated. Forexample, an operator may open or close a door by manually grasping,pulling, pushing, and/or otherwise physically interacting with the dooror an element thereof (e.g., a handle) or by operating a mechanicalcontrol (e.g., a button, toggle, spin-wheel, key, switch, cursor, screw,dial, screen, or touch-screen). In certain embodiments, a door may becontrolled by electrical or digital controls, such as by a computer. Adoor may be an automatically opening door. For example, a door mayinclude a sensor, such as a pressure, infrared, motion, or remote sensorthat detects whether the door is open or closed and/or controls when thedoor opens or closes. A door may open by mechanical, pneumatic,electrical, or other means. In some embodiments, one or more doors mayinclude one or more locking mechanisms. In particular environments, oneor more doors may include one or more interlocks (e.g., a mechanicalinterlock such as a pin, bar, or lock or an electrical interlock such asa switch) to prevent one or more doors from opening at an undesirabletime (e.g., when one or more chambers are open to the outsideenvironment).

A transfer device for moving one or more items may be used to move itemsbetween the transfer chamber and the internal chamber. In someembodiments, the transfer device includes a conveyor belt or othersimilar device for maneuvering items. Non-limiting examples of itemsthat can be moved by transfer devices include cell culture vessels,pipettes, containers, syringes, and other materials and instrumentsutilized in the culture of cells. In some embodiments, more than onetransfer device may be included. In some embodiments, one or moretransfer devices are located in the transfer chamber and/or in theinternal chamber. In some embodiments, a transfer device may include oneor more robotic elements. For example, a transfer device may include oneor more robotic arms capable of gripping, lifting, pushing, grabbing,sliding, rotating, translating, releasing, raising, lowering, and/ortilting one or more items (e.g., pipettes).

In some embodiments, the transfer device is a cell culture vesseltransfer device. As used herein, a “cell culture vessel transfer device”refers to a device that can transfer one or more cell culture vesselsfrom a first location to a second location. In some embodiments, thetransfer device is anchored within the internal chamber. In certainembodiments, the transfer device may transfer one or more items to orfrom multiple locations in an incubator cabinet. For example, a cellculture vessel transfer device may be used to move a cell culture vesselfrom a transfer chamber to an internal chamber, and/or from a storagelocation to an imaging location. In some embodiments, an incubatorcabinet includes more than one transfer device, for moving one or moreitems (e.g., separate transfer devices for transferring items betweenand within chambers). A cell culture vessel transfer device may includeone or more elements such as valves (e.g., electromagnetic or pneumaticvalves), gears, motors (e.g., electrical or stepper motors), stages(e.g., xy or xyz stages), pistons, brakes, cables, ball-screwassemblies, rack-and-pinion arrangements, grippers, arms, pivot points,joints, translational elements, or other mechanical or electricalelements. In some embodiments, a cell culture vessel transfer device mayinclude one or more robotic elements. For example, a cell culture vesseltransfer device may include a robotic arm capable of gripping, lifting,pushing, grabbing, sliding, rotating, translating, releasing, raising,lowering, and/or tilting one or more cell culture vessels. In preferredembodiments, the cell culture vessel transfer device selectively andreleasably grips one or more cell culture vessels. In certainembodiments, a cell culture vessel transfer device may include an armcoupled to a mechanical gripper. For example, an arm may include amechanical gripper at or near one end for releasably gripping a cellculture vessel and be securely coupled to a surface or element of theincubator at or near the other end. In some embodiments, a robotic armincludes a pivot point where the mechanical gripper couples to the armand one or more pivot and/or translational joints along the arm topermit flexible rotation and translation of a portion of the arm. Inthis manner, a robotic arm may access one or more cell culture vesselsat different horizontal and vertical positions within an incubatorcabinet (e.g., within a storage array in an internal chamber).

In some embodiments, a cell culture vessel transfer device is anautomated transfer device. For example, the automated transfer devicemay be a robotic arm controlled by a computer that is programmed to movecell culture vessels from a storage location within the internal chamberof the incubator to an imaging location within the internal chamber ofthe incubator. In some embodiments, a cell culture vessel transferdevice is manually operated. For example, a robotic arm located insidethe internal chamber of an incubator may be operated by auser-controlled joystick from a location outside of the internal chamberof the incubator in order to move cell culture vessels from a storagelocation within the internal chamber of the incubator to an imaginglocation within the internal chamber of the incubator.

As used herein, a “storage location” refers to a location at which oneor more cell culture vessels is stored (e.g., within an incubatorcabinet). For example, one or more cell culture vessels may be stored ata storage location and later transferred to a different location (e.g.,an imaging location). The storage location may be disposed in theinternal chamber of the incubator cabinet. A storage location may beconfigured for storing a plurality of cell culture vessels. For example,a storage location may include one or more storage arrays, racks,shelves, pigeon-holes, cubbies, trays, slots, or other positions ormechanisms. In some embodiments, a storage location may be configured tostore cell culture vessels horizontally, while in other embodiments itmay be configured to store cell culture vessels vertically. For example,a storage location may include a plurality of slots to receive cellculture vessels stacked vertically over one another. A storage locationmay be configured to hold 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 100, or any other number of cell culture vessels. Insome embodiments, a storage location may be configured to hold greaterthan 100 cell culture vessels. In some embodiments, each cell culturevessel in a storage location houses cells from a different subject. Insome embodiments, a storage location may include a mechanism for movingone or more storage arrays, racks, shelves, pigeon-holes, cubbies,trays, slots, or other positions or mechanisms. For example, a storagelocation may include one or more motors and movable stages (e.g., an xyor xyz stage) to move a storage rack from one position in an internalchamber to another position in an internal chamber, e.g., to facilitateaccess to one or more cell culture vessels stored in differentlocations. In some embodiments, the incubator cabinet may include one ormore cell culture vessel transfer devices for moving one or more cellculture vessels.

A storage location may be configured to securely hold or receive one ormore cell culture vessels. For example, one or more components of thestorage location may include one or more locking mechanisms that haveone or more adhesive, magnetic, electrical, and/or mechanical components(e.g., snaps, fasteners, locks, clasps, gaskets, o-rings, septa,springs, and other engagement members). In some embodiments, a storagelocation and/or cell culture vessel may include one or more grooves ordepressions and/or may involve pieces of molded plastic. For example, acell culture vessel may include one or more protruded features (e.g., arim or knob) that is molded for insertion into one or more correspondinggrooves, holes, or depressions at a storage location. In some cases, acell culture vessel may include one or more grooves, holes, ordepressions that are molded to fit one or more corresponding protrudedfeatures at a storage location.

As used herein, an “imager” refers to an imaging device for measuringlight (e.g., transmitted or scattered light), color, morphology, orother detectable parameters such as a number of elements or acombination thereof. An imager may also be referred to as an imagingdevice. In certain embodiments, an imager includes one or more lenses,fibers, cameras (e.g., a charge-coupled device camera or CMOS camera),apertures, mirrors, filers, light sources (e.g., a laser or lamp), orother optical elements. An imager may be a microscope. In someembodiments, the imager is a bright-field microscope. In otherembodiments, the imager is a holographic imager or microscope. In otherembodiments, the imager is a fluorescence microscope.

As used herein, a “fluorescence microscope” refers to an imaging devicewhich is able to detect light emitted from fluorescent markers presenteither within and/or on the surface of cells or other biologicalentities, said markers emitting light at a specific wavelength inresponse to the absorption a light of a different wavelength.

As used herein, a “bright-field microscope” is an imager thatilluminates a sample and produces an image based on the light absorbedby the sample. Any appropriate bright-field microscope may be used incombination with an incubator cabinet provided herein.

As used herein, a “holographic imager” is an imager that providesinformation about an object (e.g., sample) by measuring both intensityand phase information of electromagnetic radiation (e.g., a wave front).For example, a holographic microscope measures both the lighttransmitted after passing through a sample as well as the interferencepattern (e.g., phase information) obtained by combining the beam oflight transmitted through the sample with a reference beam.

A holographic imager may also be a device that records, via one or moreradiation detectors, the pattern of electromagnetic radiation, from asubstantially coherent source, diffracted or scattered directly by theobjects to be imaged, without interfering with a separate reference beamand with or without any refractive or reflective optical elementsbetween the substantially coherent source and the radiation detector(s).

In some embodiments, an incubator cabinet includes a single imager. Insome embodiments, an incubator cabinet includes two imagers. In someembodiments, the two imagers are the same type of imager (e.g., twoholographic imagers or two bright-field microscopes). In someembodiments, the first imager is a bright-field microscope and thesecond imager is a holographic imager. In some embodiments, an incubatorcabinet comprises more than 2 imagers. In some embodiments, cell cultureincubators comprise three imagers. In some embodiments, cell cultureincubators having 3 imagers comprise a holographic microscope, abright-field microscope, and a fluorescence microscope.

As used herein, an “imaging location” is the location where an imagerimages one or more cells. For example, an imaging location may bedisposed above a light source and/or in vertical alignment with one ormore optical elements (e.g., lens, apertures, mirrors, objectives, andlight collectors).

As used herein, a “fiducial mark” refers to a feature that facilitatesalignment of one or more components. In some embodiments, fiducial marksmay include one or more hole apertures over a fluorescent media orprinted or embossed fluorescent material. In other embodiments, fiducialmarks may include grids, lines, or symbols. In some embodiments, one ormore cell culture vessels include one or more fiducial marks tofacilitate alignment of one or more cell culture vessels with an imager.In some embodiments, fiducial marks may be associated with moving parts,including transfer devices and robotics devices.

In some embodiments, a cell culture vessel is substantially aligned withan imager. In some embodiments, a cell culture vessel is substantiallyaligned with an imager via the use of at least one fiducial mark. Asused herein, the term “substantially aligned” implies that one or moreelements are substantially overlapping, identical, and/or in line withone another. The substantial alignment of one or more cell culturevessels at one or more locations (e.g., imaging locations) mayfacilitate the analysis of a sample by permitting overlapping images ofthe cell culture vessel to be obtained. For example, a cell culturevessel may be imaged at a first imaging location by a first imager andsubsequently imaged at a second imaging location by a second imager. Ifthe imaging fields of the respective imagers are substantially aligned,the images recorded by the first and second imagers may be combined(“stitched together”) for analysis. One or more fiducial marks presenton one or more cell culture vessels may facilitate substantialalignment. In some cases, one or more fiducial marks present at one ormore imaging or other locations (e.g., manipulation or maintenancelocations) may facilitate substantial alignment.

As used herein, a “manipulator for manipulating cells” refers to adevice for manipulating cells in the internal chamber. The manipulatormay include one or more needles, capillaries, pipettes, and/ormicromanipulators. For example, the manipulator may include a cellpicker. A manipulator for manipulating cells may operate by detectingdesirable cells or groups thereof present at a first location based on apredetermined criterion and transferring the desired cells or groupsthereof from the first location to a second location. A cell picker maydetect, pick, and/or transfer desirable or undesirable (e.g.,pre-differentiated cell weeding) cells or groups thereof based on amanual or automated analysis. In some embodiments, information producedby an imager may be analyzed to detect desirable or undesirable cells.The cell picker may then transfer the desirable or undesirable cells tothe second location. For example, an imager may image cells in or on acell culture vessel at an imaging location, and the image used toidentify desirable or undesirable cells or groups thereof. The cellpicker may then transfer the desirable or undesirable cells, e.g., bycontacting each desired cell or cells with a needle, capillary, pipette,or micromanipulator and effecting a movement of the cell or cells, fromtheir first location to a second location in or on the cell culturevessel or elsewhere in the internal chamber. In some embodiments, thefirst location of the cells may be in or on a cell culture vessel. Inparticular embodiments, a cell picker transfers cells from a firstlocation in or on a cell culture vessel to a second location on the samecell culture vessel. In other embodiments, a cell picker transfers cellsfrom a first location in or on a first cell culture vessel to a secondlocation in or on a second cell culture vessel. In certain otherembodiments, a cell picker transfers cells from a first location in oron a cell culture vessel to a second location in the internal chamberthat is not in or on a cell culture vessel.

In some embodiments, the manipulator includes at least onemicroelectrode. As used herein, the term “microelectrode” refers to anelectrical conductor used to deliver electrical stimulation to a cell.For example, microelectrodes can be used to deliver genetic materialinto a cell by electroporation. In some embodiments, the manipulatorincludes at least one microinjector. Generally, microinjectors are glassmicropipettes that have been pulled to form a sharp, hollow structurecapable of piercing the membrane of a cell and serving as a conduit forthe introduction of genetic material into the cell. In some embodiments,cell cultures are manipulated in other ways during culture in incubatorsand vessels described herein. For example, cell cultures may betransfected with nucleic acids (e.g., DNA or RNA) or exposed to viralinfection (e.g., using recombinant virus particles to deliver DNA orRNA).

In some embodiments, a manipulator includes fluid handling devices. Forexample, a manipulator may include one or more liquid dispensingapparatus, such as pipette tip holders or a cell printing device. Insome embodiments fluid handling devices are automated. In some aspects,manipulators having automated fluid handling systems that dispensegrowth media from fluid storage vessels located inside the internalchamber of the incubator to cell culture vessel can be used.

In some embodiments (e.g., for adherent cell cultures), culture mediacan be removed directly by aspiration and replaced with fresh media. Insome embodiments (e.g., for non-adherent/suspension cultures), mediachanges can involve centrifuging a cell culture, removing the oldculture media and replacing it with fresh media. In some embodiments,the centrifuge is located in the internal chamber of an incubator. Insome embodiments, culture vessels allow for continuous mediareplacement. In some embodiments, the incubators described herein mayinclude one or more components that can be used to process, replace,supply, and/or maintain different aspects of a culture media to supportcells. Incubators may include a reservoir containing waste media and/ora reservoir containing fresh media. Such reservoirs may be present(e.g., for temporary storage) within a refrigerator inside the incubatoror a refrigerated section of the incubator. In some embodiments, one ormore reservoirs are provided outside the incubators and piping isprovided into and out from the incubator space to supply or draw from aliquid handler units (e.g., liquid handle units having an aspirator) ortemporary reservoir within the incubator to facilitate cells feeding,media changes, and other related needs. For suspension cells, devicesmay be provided within the incubator to separate cells from waste media(e.g., centrifuge(s) to facilitate cell pelleting) to facilitateautomated media changes as part of an incubator provided herein. In someembodiments, the document provides a system comprising a cell cultureincubator connected to a computer, capable of automatically monitoringand adjusting cell culture conditions for optimal growth of the cellculture.

In some embodiments, cells are passaged within an incubator describedherein. In some embodiments, a cell culture is split, and a subset ofthe cell culture is transferred to a fresh culture vessel for furthergrowth. In some embodiments (e.g., for adherent cell cultures), cellsare detached (e.g., mechanically, for example, using gentle scraping,and/or enzymatically, for example, using trypsin-EDTA or one or moreother enzymes) from a surface prior to being transferred to a freshculture vessel. In some embodiments (e.g., for suspension cellcultures), a small volume of a cell culture is transferred to a freshculture vessel.

In some embodiments, a manipulator is manually operated. For example, amanipulator having a fluid handling system located inside the internalchamber of an incubator cabinet may be electronically-linked to andcontrolled by a user-directed joystick located outside the internalchamber of the incubator cabinet. In some embodiments, the user-directedjoystick is connected to a display device. In some embodiments, thedisplay device shows images captured by an imaging device inside theinternal chamber of the incubator cabinet.

In some embodiments, a manipulator is automated. For example, amanipulator inside an internal chamber of an incubator cabinet may beelectronically connected to a controller outside of the incubatorcabinet that directs the manipulator. In some embodiments, the computerautomatically remembers where particular cell culture vessels arelocated inside the incubator. In some embodiments, the computer usesbarcodes or other identifying information to verify that the saidlocations are correctly remembered.

One or more elements of the manipulator for manipulating cells may besterilized, for example using a sterilizing composition or method (e.g.,ethanol or ozone gas), prior to manipulation.

As used herein, “manipulation location” refers to the location at whichcells are manipulated by a manipulator for manipulating cells (e.g., acell picker). In certain embodiments, the manipulation location may bethe same as the imaging location.

According to one aspect, the cell culture incubator includes anincubator cabinet with an imaging location and a manipulating location.Cells of a cell culture vessel are imaged at the imaging location by animager and manipulated at the manipulating location by a manipulator. Insome embodiments, the imaging location and the manipulating location aretwo distinct locations within the incubator cabinet. The cell cultureincubator may include a transfer device that moves cell culture vesselsbetween the imaging location and the storage location. In otherembodiments, the imaging location and the manipulating location are thesame, such that the cells of culture vessels are imaged at themanipulation location.

In some embodiments, an imager may be used in conjunction with amanipulator. For example, an imager may image cells in or on a cellculture vessel at an imaging location, and the image used to identifydesirable cells or groups thereof. The manipulator may then transfer thedesirable cells, e.g., by contacting each desired cell or cells with aneedle, capillary, pipette, or micromanipulator and effecting a movementof the cell or cells, from their first location to a second location inor on the cell culture vessel or elsewhere in the internal chamber. Insome embodiments, the manipulator aseptically transfers growth media,growth factors, or expression vectors into cell culture vessels.

In some embodiments, a single location within the incubator cabinet mayserve as an imaging location and a manipulating location. In someembodiments, an imaging location and a manipulating location are atdifferent locations within the incubator cabinet. In one embodiment,cells are imaged as they are manipulated by the manipulator.

In some embodiments, the environment inside an incubator is controlledby a control system that may be configured to control the temperature,humidity, carbon dioxide, oxygen and other gaseous components ((e.g.,sterilization gases, such as, ozone, and hydrogen peroxide)) inside theincubator (e.g., in one or more internal chambers). In some embodiments,a control system controls the environmental conditions (e.g.,temperature, humidity, carbon dioxide, oxygen and other gaseouscomponents) within each internal chamber separately. For example, inorder to protect sensitive mechanical, electronic and opticalcomponents, the humidity of an internal chamber may be maintained at alower level than an internal chamber having a storage location. In someembodiments, the incubator is further provided with a monitoring systemwith predefined sensors. Examples of monitoring devices include but arenot limited to oxygen monitors, carbon dioxide monitors, ozone gasdetectors, hydrogen peroxide monitors and multi gas monitors. Forexample, in some embodiments, an incubator advantageously includes aplurality of sensors responsive to different parameters relevant to cellgrowth, which may include temperature, air purity, contaminant levels,pH, humidity, N₂, CO₂, O₂ and light. By means of this monitoring system,parameters in the incubator can be measured using sensors for theduration of a culture or process. In some embodiments, parametersmeasured by the sensors are transmitted by the monitoring system via aline to a computer-controlled monitoring and control system for furtherprocessing as discussed elsewhere herein.

In some embodiments, an environmental monitoring system can be used inconjunction with an incubator described herein. In some embodiments, oneor more sensors that provide for the measurement of temperature, aircomposition (e.g., CO₂ concentration, O₂ concentration, etc.), and/orhumidity of the system can be associated with an incubator (e.g., fittedwithin an incubator cabinet). In some embodiments, one or more suchsensors can be incorporated as part of an incubator (e.g., attached to,integral to, or otherwise connected to an internal wall or door of theincubator). In some cases, one or more sensors can be positioned at anysuitable location(s) outside or inside an incubator cabinet (e.g.,within a transfer chamber and/or an internal chamber, for exampleattached to an internal wall, and/or upper or lower internal surface).

In some embodiments, a gas sensor is provided that can provide a readingin real time of the concentration of gas in contact with the sensor(e.g., gas in a cabinet, or ambient air) in percent, parts per million,or any other standard unit. Gas sensors for use in the methods andincubators provided herein include CO₂ sensors, O₂ sensors, N₂ sensors,ozone gas detectors, hydrogen peroxide monitors, multi gas monitors, andCO sensors. Such sensors are available from a number of commercialsources. In some cases, the environment of the incubator may bemodulated or controlled based upon the information provided by thesensors described herein. For example, the level of CO₂ in an incubatormay be increased upon indication from a CO₂ sensor that a lower thandesirable concentration of CO₂ is present in the incubator.

In some embodiments, one or more heating or cooling elements can beincorporated within the incubator (e.g., on an inner surface of thecabinet or door, and/or integrated within one or more of the wallsand/or the base of the cabinet) for purposes of controlling thetemperature within the incubator. In some embodiments, a heating elementcan be used for thawing liquids, for example, cell culture media orother reagents.

In some embodiments, one or more air or oxygen sources, carbon filters,and/or one or more humidification or dehumidification systems areconnected to the incubator and configured to control the level ofoxygen, carbon dioxide, and/or humidity within the incubator (e.g., inresponse to signals from the one or more sensors in or attached to theincubator). In some embodiments, one or more controllers are attached tothe sensors and other systems to control the internal environment of theincubator.

In some embodiments, an incubator can include one or more light sources(e.g., an incandescent bulb, LED, UV, or other light source). These canbe placed within the incubator to illuminate regions within the cabinet.In some embodiments, the culture system operation is monitored using acamera or other light sensitive device that can be placed within oroutside the incubator. In some embodiments, the light source is asterilizing light source. For example, a UV lamp may be located withinthe transfer chamber and/or the interior chamber of an incubatorprovided herein.

In some embodiments, the incubator includes a transparent object (e.g.,window) that allows visible light or other light wavelengths from withinthe incubator to be detected by a camera or other light sensitive deviceplaced outside the incubator. In some embodiments, the inner surface ofthe transparent object can be wiped (e.g., from the inside of thecabinet) to prevent or remove condensation droplets that may accumulate(e.g., due to the humid air inside the incubator) on the inner surfaceand interfere with the monitoring of the system. In some embodiments,the surface can be wiped by a wiper that is automatically controlled bya controller.

In some embodiments, a sensor or other feature is provided to detectwhen one or more doors of an incubator are opened (e.g., when anincubator cabinet door, such as an external or internal door, isopened). Such features are useful because they allow operators to keeptrack of or be warned of any unscheduled or unauthorized openings of theincubator (e.g., the incubator cabinet) that could jeopardize sterility,spoil a production, compromise an assay or experiment, etc.

In some embodiments, a radiofrequency beacon or other signal source islocated within the incubator (e.g., within the incubator cabinet) thatcan be used to determine the location of one or more devices within theincubator cabinet (e.g., devices having sensors that can detect thesignal and use it to determine their location). In some embodiments, thedevices could have signal sources and the sensor(s) could be locatedwithin one or more of the chambers of an incubator cabinet (e.g.,located on an internal surface of an internal chamber).

In some embodiments, light signals or lasers (e.g., a grid of lasersignals) can be used to determine the location and/or identity of one ormore devices or components within the incubator cabinet. Suchinformation can be communicated, e.g., wired or wirelessly, to anexternal computer or monitoring station. The information can be used tocontrol operation of a transfer device, e.g., a robotic arm, within theincubator cabinet to ensure that the transfer device can grab,manipulate, or maneuver devices or items appropriately within theincubator cabinet.

In some embodiments, before containers or vessels are brought into anincubator cabinet, a user can select an automation system protocol basedon the particular containers, vessels, ingredients, or cells that arebeing inserted into the incubator cabinet. Relevant information relatedto the incubator and/or one or more incubator components, and the cellsbeing grown can be entered into a data system. For example, one or moreidentifiers such as barcodes (e.g., 1D or 2D barcodes) can be placed onthe container or vessel and other significant information, such as, thetype of container, the contents of the container and, what assays ormanipulations are to be performed on the sample in the container can bespecified. In some embodiments, information related to the incubatorsystem and/or cells can be contained in one or more barcodes, on aseparate data system, or a combination thereof. The user may also enterinformation that identifies the dimensionality (e.g., height, diameter)of the vessel or other container or the system itself determine measurethe height of the vessel or other container. Using this information, therobotic arm may be requested to transport a particular container, suchas when an analytical module is ready to perform an assay or othermanipulation on cells grown in the vessels or has completed performingan assay or manipulation.

Computer and Control Equipment

The incubators provided herein include several components, includingsensors, environmental control systems, robotics, etc. which may operatetogether at the direction of a computer, processor, microcontroller orother controller. The components may include, for example, a transferdevice (e.g., robotic arm), a liquid handling devices, a delivery systemfor delivering culture vessels, or other components to or from theincubator cabinet, an environmental control system for controlling thetemperature and other environmental aspects of the incubator cabinet, adoor operation system, an imaging or detection system, and a cellculture assay system.

In some cases, operations such as controlling operations of a cellculture incubator and/or components provided therein or interfacingtherewith may be implemented using hardware, software or a combinationthereof. When implemented in software, the software code can be executedon any suitable processor or collection of processors, whether providedin a single component or distributed among multiple components. Suchprocessors may be implemented as integrated circuits, with one or moreprocessors in an integrated circuit component. A processor may beimplemented using circuitry in any suitable format.

A computer may be embodied in any of a number of forms, such as arack-mounted computer, a desktop computer, a laptop computer, or atablet computer. Additionally, a computer may be embedded in a devicenot generally regarded as a computer but with suitable processingcapabilities, including a Personal Digital Assistant (PDA), a smartphone or any other suitable portable, mobile or fixed electronic device,including the incubator itself.

In some cases, a computer may have one or more input and output devices.These devices can be used, among other things, to present a userinterface. Examples of output devices that can be used to provide a userinterface include printers or display screens for visual presentation ofoutput and speakers or other sound generating devices for audiblepresentation of output. Examples of input devices that can be used for auser interface include keyboards, and pointing devices, such as mice,touch pads, and digitizing tablets. In other examples, a computer mayreceive input information through speech recognition or in other audibleformat, through visible gestures, through haptic input (e.g., includingvibrations, tactile and/or other forces), or any combination thereof.

One or more computers may be interconnected by one or more networks inany suitable form, including as a local area network or a wide areanetwork, such as an enterprise network or the Internet. Such networksmay be based on any suitable technology and may operate according to anysuitable protocol and may include wireless networks, wired networks, orfiber optic networks.

The various methods or processes outlined herein may be coded assoftware that is executable on one or more processors that employ anyone of a variety of operating systems or platforms. Such software may bewritten using any of a number of suitable programming languages and/orprogramming or scripting tools, and may be compiled as executablemachine language code or intermediate code that is executed on aframework or virtual machine.

One or more algorithms for controlling methods or processes providedherein may be embodied as a readable storage medium (or multiplereadable media) (e.g., a computer memory, one or more floppy discs,compact discs (CD), optical discs, digital video disks (DVD), magnetictapes, flash memories, circuit configurations in Field Programmable GateArrays or other semiconductor devices, or other tangible storage medium)encoded with one or more programs that, when executed on one or morecomputers or other processors, perform methods that implement thevarious methods or processes described herein.

In some embodiments, a computer readable storage medium may retaininformation for a sufficient time to provide computer-executableinstructions in a non-transitory form. Such a computer readable storagemedium or media can be transportable, such that the program or programsstored thereon can be loaded onto one or more different computers orother processors to implement various aspects of the methods orprocesses described herein. As used herein, the term “computer-readablestorage medium” encompasses only a computer-readable medium that can beconsidered to be a manufacture (e.g., article of manufacture) or amachine. Alternatively or additionally, methods or processes describedherein may be embodied as a computer readable medium other than acomputer-readable storage medium, such as a propagating signal.

The terms “program” or “software” are used herein in a generic sense torefer to any type of code or set of executable instructions that can beemployed to program a computer or other processor to implement variousaspects of the methods or processes described herein. Additionally, itshould be appreciated that according to one aspect of this embodiment,one or more programs that when executed perform a method or processdescribed herein need not reside on a single computer or processor, butmay be distributed in a modular fashion amongst a number of differentcomputers or processors to implement various procedures or operations.

Executable instructions may be in many forms, such as program modules,executed by one or more computers or other devices. Generally, programmodules include routines, programs, objects, components, datastructures, etc. that perform particular tasks or implement particularabstract data types. Typically, the functionality of the program modulesmay be combined or distributed as desired in various embodiments.

Also, data structures may be stored in computer-readable media in anysuitable form. Non-limiting examples of data storage include structured,unstructured, localized, distributed, short-term and/or long termstorage. Non-limiting examples of protocols that can be used forcommunicating data include proprietary and/or industry standardprotocols (e.g., HTTP, HTML, XML, JSON, SQL, web services, text,spreadsheets, etc., or any combination thereof). For simplicity ofillustration, data structures may be shown to have fields that arerelated through location in the data structure. Such relationships maylikewise be achieved by assigning storage for the fields with locationsin a computer-readable medium that conveys relationship between thefields. However, any suitable mechanism may be used to establish arelationship between information in fields of a data structure,including through the use of pointers, tags, or other mechanisms thatestablish relationship between data elements.

In some embodiments, information related to the operation of theincubator (e.g., temperature, humidity, gas composition, images, cellculture conditions, etc., or any combination thereof) can be obtainedfrom one or more sensors associated with the incubator (e.g., locatedwithin the incubator cabinet, or located within the incubator butoutside the incubator cabinet), and can be stored in computer-readablemedia to provide information about conditions during a cell cultureincubation. In some embodiments, the readable media comprises adatabase. In some embodiments, said database contains data from a singleincubator. In some embodiments, said database contains data from aplurality of incubators. In some embodiments, data is stored in a mannerthat makes it tamper-proof. In some embodiments, all data generated bythe instrument (e.g., an incubator) is stored. In some embodiments, asubset of data is stored.

In some embodiments, the component (e.g., a computer) controls variousprocesses performed inside the incubator. For example, a computer maydirect control equipment (e.g., a manipulator, an imager, a fluidhandling system, etc.). In some embodiments, the computer controlsimaging of cell cultures, picking of cells, weeding of cells (e.g.,removal of cell clumps), monitoring of cell culture conditions,adjustment of cell culture conditions, tracking of cell culture vesselmovement within the incubator, and/or scheduling of any of the foregoingprocesses.

Turning to the figures, FIG. 1 shows a schematic of an illustrativeembodiment of a cell culture system. In some embodiments, a cell culturesystem includes an incubator cabinet having an external door (100) whichopens into an internal chamber (101). Inside the internal chamber are aplurality cell culture vessels (102), each vessel having a passageconfigured to permit materials to be aseptically passed into or out fromthe vessel; each cell culture vessel (P1, P2, P3, P4, P5, and P6) maycontain a cell sample from a different subject.

FIGS. 2A-2B show schematics of illustrative embodiments of a cellculture systems. FIG. 2A depicts a cell culture system including anincubator cabinet having an internal chamber (101) housing a pluralityof cell culture vessels (102), each vessel having a passage configuredto permit materials to be aseptically passed into or out from thevessel; an internal door (103), which opens into a transfer chamber(104), and forms a gas-tight seal when closed; a cell culture vesseltransfer device (105); and, an external door (100) that connects thetransfer chamber to the exterior of the incubator cabinet when open.FIG. 2B depicts a schematic of the cell culture system embodied in FIG.2A, further including an ozone generator (106) and a pump (107). In someembodiments, the ozone generator and pump are in fluid communication. Insome embodiments, the ozone generator and the incubator cabinet are influid communication. For example, the ozone generator can be in fluidcommunication with the internal chamber and/or the transfer chamber ofthe incubator cabinet via a pipe or tubing connected to an inlet oroutlet of the incubator cabinet. In some embodiments, the pump and theincubator cabinet are in fluid communication. For example, the pump canbe in fluid communication with the internal chamber and/or the transferchamber of the incubator cabinet via a pipe or tubing connected to aninlet or outlet of the incubator cabinet. In some embodiments, the pumpis used to remove ozone gas from the internal chamber and/or transferchamber of the incubator cabinet.

FIG. 3 shows a schematic of an illustrative embodiment of a cell culturesystem. In some embodiments, the cell culture system includes anincubator cabinet having an external door (100), which opens into atransfer chamber (104). The transfer chamber has two internal doors(103, 108). The first internal door (103) opens into a first internalchamber (101), which houses a plurality of cell culture vessels, eachvessel having a passage configured to permit materials to be asepticallypassed into or out from the vessel. The second internal door (108) opensinto a second internal chamber (109). The second internal chamberincludes an imager (110), a manipulator (111) and a manipulationlocation (112). In some embodiments, the second internal chamber alsoincludes a fluid reservoir (113) and/or a centrifuge (114). In someembodiments, the second internal chamber includes a FACS machine.

FIG. 4 shows a schematic of an illustrative embodiment of a cell culturesystem. In some embodiments, the incubator cabinet includes an externaldoor (100) that opens into a first internal chamber (101), which housesa plurality of cell culture vessels, each vessel configured to permitmaterials to be aseptically passed into or out from the vessel, theincubator cabinet includes a transfer device (105). An internal doorconnects the first internal chamber to a second internal chamber (109),that includes an imager (110).

The above aspects and embodiments may be employed in any suitablecombination, as the present invention is not limited in this respect.

It should be understood that aspects of the invention are describedherein with reference to certain illustrative embodiments and thefigures. The illustrative embodiments described herein are notnecessarily intended to show all aspects of the invention, but ratherare used to describe a few illustrative embodiments. Thus, aspects ofthe invention are not intended to be construed narrowly in view of theillustrative embodiments. In addition, it should be understood thataspects of the invention may be used alone or in any suitablecombination with other aspects of the invention.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art. Such alterations, modifications, and improvements are intendedto be part of this disclosure, and are intended to be within the spiritand scope of the invention. Accordingly, the foregoing description anddrawings are by way of example only.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

What is claimed is:
 1. A method for producing a mammalian cell culturecomprising the steps of: providing a cell culture vessel comprising apassage configured to permit materials to be aseptically transferredinto or out from the vessel; introducing a mammalian cell sample intosaid cell culture vessel through said passage; placing said cell culturevessel into a sterile internal chamber of an incubator; and, expandingthe cell sample in said sterile internal chamber, thereby producing amammalian cell culture.
 2. The method of claim 1, wherein said passageis pierceable, self-sealing membrane.
 3. The method of claim 1 or 2,wherein said passage is covered by a gas-permeable membrane.
 4. Themethod of claim 1, wherein said passage is a sterile, disposable tube.5. The method of claim 1, wherein said passage is a network ofmicrofluidic channels.
 6. The method of any one of claims 1 to 5,further comprising aseptically introducing growth media through saidpassage in the vessel.
 7. The method of any one of claims 1 to 5,further comprising aseptically introducing a biological material throughsaid passage in the vessel.
 8. The method of claim 7, wherein thebiological material is a cell growth factor.
 9. The method of claim 7,wherein the biological material is a nucleic acid molecule.
 10. Themethod of claim 9, wherein the nucleic acid molecule is a nucleic acidvector.
 11. The method of claim 10, wherein the nucleic acid vector is atransfection vector.
 12. The method of claim 10, wherein the nucleicacid vector is a transduction vector.
 13. The method of claim 10,wherein the nucleic acid vector comprises transgenic material.
 14. Themethod of any one of claims 1 to 3, further comprising asepticallymonitoring conditions of the growth media.
 15. The method of any one ofclaims 1 to 3, further comprising maintain a constant temperature rangein the incubator.
 16. The method of any one of claims 1 to 3, whereinthe incubator is maintained at about 37 degrees Celsius.
 17. The methodof any one of claims 1 to 3, further comprising aseptically monitoringconditions of the cell.
 18. The method of any one of claims 1 to 3,further comprising aseptically imaging the cell culture.
 19. The methodof any one of claims 1 to 3, further comprising filtering the cellculture.
 20. The method of any one of claims 1 to 3, further comprisingaseptically removing an aliquot of the cell culture.
 21. The method ofany one of claims 1 to 20, wherein the incubator is a cell culturesystem as described in any one of claims 22 to
 49. 22. A cell cultureincubator comprising: a transfer chamber; one or more internal chambers;an external door opening from an external environment to the transferchamber; a first internal door opening from the transfer chamber to afirst internal chamber; a second internal door opening from the transferchamber to a second internal chamber; and, a transfer device for movingone or more items between the transfer chamber and the first internalchamber and/or between the transfer chamber and the second internalchamber and/or between the second internal chamber and the firstinternal chamber.
 23. The cell culture incubator of claim 22 furthercomprising an ozone generator coupled to a pump, wherein the ozonegenerator is configured for supplying ozone gas to the transfer chamber.24. The cell culture incubator of claim 23, wherein the ozone generatoris configured for supplying ozone gas to internal chamber.
 25. The cellculture incubator of any one of claims 22 to 24, wherein the externaldoor forms a substantially gas-tight seal when closed.
 26. The cellculture incubator of any one of claims 22 to 25, wherein the firstinternal door and the second internal door each forms a substantiallygas-tight seal when closed.
 27. The cell culture incubator of any one ofclaims 23 to 26, wherein the pump is configured to remove ozone gas fromthe transfer chamber and/or one or more internal chamber.
 28. The cellculture incubator of any one of claims 22 to 27, wherein the internalchamber comprises a storage location.
 29. The cell culture incubator ofclaim 28, wherein the storage location is configured to hold a pluralityof cell culture vessels.
 30. The cell culture incubator of claim 29,wherein each vessel of the plurality of cell culture vessels is taggedwith a unique barcode.
 31. The cell culture incubator of any one ofclaims 22 to 30, wherein the internal chamber comprises an imager and animaging location.
 32. The cell culture incubator of any one of claims 22to 31, wherein the imager is a holographic imager.
 33. The cell cultureincubator of any one of claims 22 to 31, wherein the imager is amicroscope, optionally a bright-field microscope or a fluorescencemicroscope.
 34. The cell culture incubator of any one of claims 31 to33, further comprising a controller for the imager.
 35. The cell cultureincubator of any one of claims 22 to 34, wherein the internal chambercomprises a manipulator and a manipulation location.
 36. The cellculture incubator of claim 35, wherein the manipulator is a cell picker.37. The cell culture incubator of claim 35, wherein the manipulatorcomprises a fluid handling system.
 38. The cell culture incubator of anyone of claims 35 to 37, further comprising a controller for themanipulator.
 39. The cell culture incubator of any one of claims 22 to38, wherein the internal chamber comprises a fluid storage location. 40.The cell culture incubator of any one of claims 22 to 39, wherein theinternal chamber comprises a cell sorting or cell isolation apparatus.41. The cell culture incubator of claim 40, wherein the cell sorting orcell isolation apparatus is a centrifuge.
 42. The cell culture incubatorof claim 40, wherein the cell sorting or cell isolation apparatus is aFluorescence-Activated Cell Sorting (FACS) machine.
 43. The cell cultureincubator of any one of claims 22 to 42, wherein the transfer devicecomprises one or more robotic elements.
 44. The cell culture incubatorof any one of claims 22 to 43, further comprising a controller for thetransfer device.
 45. The cell culture incubator of any one of claims 34to 44, wherein the controller for the imager, the controller for themanipulator and/or the controller for the transfer device are externalto the incubator cabinet.
 46. The cell culture incubator of claim 45,wherein the controller for the imager, the controller for themanipulator and/or the controller for the transfer device comprise acomputer.
 47. The cell culture incubator of claim 46, wherein a singlecomputer controls the imager, the manipulator and/or the transferdevice.
 48. The cell culture incubator of any one of claims 22 to 47,further comprising a barcode scanner.
 49. The cell culture incubator ofclaim 48, wherein the barcode scanner is connected to a computer,wherein the computer is external to the incubator cabinet.
 50. A cellculture system comprising 2 or more cell culture vessels, wherein eachvessel comprises cells from a different patient and wherein each vesselcomprises a passage configured to permit materials to be asepticallypassed into or out from the vessel.